The enzyme sulfhydryl oxidase (SHX) catalyzes--in the presence of oxygen--the conversion of thiol compounds to their corresponding disulfides according to the equation: EQU 2RSH+O.sub.2 .fwdarw.RSSR+H.sub.2 O.sub.2
Several enzymes with the ability to catalyze this reaction, derived from both mammalian and microbial sources, have been reported in the scientific literature. In 1956, Mandels reported that the spore s of the fungus Myrothecium varrucaria contained a sulfhydryl oxidase which catalyzed the oxidation of reduced glutathione (GSH), cysteine and homocysteine with concomitant reduction of O.sub.2 to H.sub.2 O.sub.2. Thiooxidase activity was found in the cell-free culture filtrates of the fungi Piricularia oryzae and Polyporous versicolor (Neufeld et al., 1950); this oxidase failed to utilize GSH and cysteine as substrates, but oxidized ethylenic linked thiol groups in a wide range of compounds. Aurbach et al. reported that thiooxidase isolated from P. oryzae (Aurbach and Jakoby, 1962) was responsible for the metapolyphenol oxidase activity that had been observed in crude P. oryzae extracts. In 1975, Olsen isolated a sulfhydryl oxidase from the culture fluid of an organism believed to be Dactylium dendroides. This copper metalloenzyme was found in mycelium extracts which also contained galactose oxidase. Olsen reported that the purified sulfhydryl oxidase was capable of reactivating reductively denatured galactose oxidase, as well as protecting the latter enzyme against inactivation by allow molecular weight inhibitor present in the culture fluid.
In 1972, Young and Nimmo reported that an impure preparation of glucose oxidase derived from Spergillus niger catalyzed the oxidation of GSH, and speculated that the impure glucose oxidase preparation may contain an enzyme which catalyzes this reaction.
Sulfhydryl oxidases have also been obtained from mammalian sources. In 1975 Janolino and Swaisgood purified an iron-dependent sulfhydryl oxidase from bovine milk which demonstrated activity toward GSH, cysteine, dithiothreitol, 2-mercaptoethanol and reduced ribonuclease A (Janolino and Swaisgood, 1975). The enzyme also restored reduced chymotrypsinogen to a form capable of undergoing conversion to active chymotrypsin (Janolino et al., 1978), and catalyzed the conversion of xanthine dehydrogenase to xanthine oxidase (Clare et al., 1981). Other researchers report that milk extracts from other sources, including human, (Isaacs et al. 1981) as well as kidney homogenates and mammalian pancreas tissue (Clare et al. 1984) exhibit sulfhydryl oxidase activity. Takamuri reported that an enzyme isolated from the skin of young rats catalyzed the oxidation of dithiothreitol, dithioerythritol, D-penicillamine and L-cysteine, although GSH and 2-mercaptoethanol were very poor substrates (Takamuri et al., 1980).
There is no evidence that any of the aforementioned enzymes are flavoproteins. However, a sulfhydryl oxidase activity discovered in rat epididymal fluid (Chang and Morton, 1975) was later demonstrated to be the activity of an FAD-dependent (flavin adenine dinocleotide) enzyme (Ostrowski et al, 1979; Ostrowski and Kistler, 1980). The best substrates for this sulfhydryl oxidase were reported to be dithiothreitol, GSH and cysteine; as in the case of the skin and bovine milk enzymes, this enzyme was capable of reactivating reduced ribonuclease A.
The precise biochemical role of SHX has not yet been determined. Janolino and Swaisgood (1975) suggest that the enzyme may contribute to the formation of disulfide bonds during or after the cellular synthesis of proteins. Chang and Morton (1975) suggest that SHX may protect other enzymes or biological tissues against adventitious reductive damage.
Although the commercial applications of SHX are quite broad and include any context where the oxidation of free sulfhydryl groups to sulfide bonds is desirable, SHX has heretofore been unavailable in quantities of sufficient size and purity for commercial usage. In particular, the expense and availability of mammalian tissue make mammalian sources of SHX unlikely candidates for production of SHX on a large and economically efficient scale. Microbial sources of SHX, on the other hand, could potentially produce SHX on a large scale in a relatively inexpensive and efficient manner. It has now been discovered that A. niger produces SHX in sufficient amounts to make it an excellent source of SHX on a commercial scale. A. niger-derived SHX is a flavoprotein enzyme which catalyzes the conversion of low molecular weight thiol compounds to their corresponding disulfides and is also capable of accepting protein associated thiol groups as substrates.